Methods for treating cancer

ABSTRACT

The present invention relates to methods of treating patients with advanced forms of cancer, such as clear cell renal cell carcinoma, in which X4P-001 is administered in order to reduce angiogenic escape that typically occurs with TKI therapy. The methods demonstrate surprising results, including regression of tumor size and cell number, with comparatively little toxicity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/267,052, filed Dec. 14, 2015, theentirety of which is hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant CA101942awarded by the National Institutes of Health. The government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods for treating cancer, inparticular, methods for overcoming resistance to treatment with VEGF-Rantagonists in cancers, such as renal cell carcinoma.

BACKGROUND OF THE INVENTION

In ˜75% of patients with sporadic clear-cell renal cell carcinoma(ccRCC) there is functional loss of the VHL gene, typically by mutation,but also silencing by hypermethylation. VHL encodes the vonHippel-Lindau tumor suppression protein, which mediates proteolyticdegradation of the hypoxia-inducible factor (HIF)-α [2]. Loss of thisfunction results in increased levels of HIF-α, increased expression ofVEGF, tumor angiogenesis, and, ultimately, the hypervascularitycharacteristic of these malignancies. Multiple agents that block theactivation of the VEGF pathway have been shown to improve outcomes,including tyrosine kinase inhibitors (TKIs), such as sunitinib,axitinib, sorafenib or pazopanib, that block the VEGF signaling pathwayand bevacizumab, a monoclonal antibody, that binds circulating VEGF andthus prevents the ligand from binding to the VEGF receptor.

Despite the demonstrated benefits of such angiogenesis inhibitors inccRCC, the approach is not curative. Although many patients respondinitially, most of them experience relapse and progression. There is aclear unmet need for agents that improve outcomes by preventing ordelaying treatment resistance.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrates the increase in tumor regression observed intwo murine models of tumor xenografts that have been treated with acombination of X4P-001 and axitinib, as described in Example 1. FIG. 1Ashows the relative effects on tumor volume of treatment of murine 786-0xenografts in mice treated with control, axitinib or X4P-001 as singleagents, and with a combination of X4P-001 and axitinib. FIG. 1B showsthe relative effects on tumor volume of treatment of murine A498xenografts with control, axitinib or X4P-001 as single agents, and witha combination of X4P-001 and axitinib. Treatment was initiated whentumor nodules reached ˜7 mm mean diameter.

FIGS. 2A-2D illustrate the increase in tumor cell death observed in themurine 786-0 xenograft model described in Example 1 in mice treated withcontrol (FIG. 2A), axitinib (FIG. 2B) or X4P-001 (FIG. 2C) as singleagents, or with a combination of X4P-001 and axitinib (FIG. 2D).

FIG. 3A-3D illustrate the increase in tumor cell death observed in themurine 498 xenograft model described in Example 1 in mice treated withcontrol (FIG. 3A), axitinib (FIG. 3B) or X4P-001 (FIG. 3C) as singleagents, or with a combination of X4P-001 and axitinib (FIG. 3D).

FIGS. 4A-4D illustrate the decreased presence of Ki-67+ and CD34+ cellsobserved in two murine models of tumor xenografts described in Example 1that have been treated with a combination of X4P-001 and axitinib. FIG.4A shows the relative preponderance of expression of Ki-67 by tumorcells in the murine 786-0 xenograft model after treatment with control,axitinib or X4P-001 as single agents, and with a combination of X4P-001and axitinib. FIG. 4B shows the relative preponderance of expression ofCD34 by tumor cells in the murine 786-0 xenograft model after treatmentwith control, axitinib or X4P-001 as single agents, and with acombination of X4P-001 and axitinib. FIG. 4C shows the relativepreponderance of expression of Ki-67 by tumor cells in the murine A498xenograft model after treatment with control, axitinib or X4P-001 assingle agents, and with a combination of X4P-001 and axitinib. FIG. 4Dshows the relative preponderance of expression of CD34 by tumor cells inthe murine A498 xenograft model after treatment with control, axitinibor X4P-001 as single agents, and with a combination of X4P-001 andaxitinib. In all instances, the reduction in expression of Ki-67 andCD34 was significantly reduced (p<0.05) in mice treated with thecombination compared to mice treated with X4P-001.

FIGS. 5A-5D illustrate the significantly reduced MDSC infiltrationobserved in two murine models of tumor xenografts described in Example 1in mice that have been treated with a combination of X4P-001 andaxitinib. FIG. 5A shows the relative reduction in area of MDSCinfiltration in xenografts in the murine 786-0 xenograft model aftertreatment with control, axitinib or X4P-001 as single agents, and with acombination of X4P-001 and axitinib. FIG. 5B shows the relativereduction in area of MDSC infiltration in xenografts in the murine A498xenograft model after treatment with control, axitinib or X4P-001 assingle agents, and with a combination of X4P-001 and axitinib. FIG. 5Cshows the relative number of MDSC (CD11b+ GR-1+) cells infiltratingxenografts in the murine 786-0 xenograft model after treatment withcontrol, axitinib or X4P-001 as single agents, and with a combination ofX4P-001 and axitinib. FIG. 5D shows the relative number of MDSC (CD11b+GR-1+) cells infiltrating xenografts in the murine A498 xenograft modelafter treatment with control, axitinib or X4P-001 as single agents, andwith a combination of X4P-001 and axitinib.

FIG. 6 and FIG. 7 illustrate through immunofluorescence (IF) the MDSC(CD11b+ GR-1+) infiltrating 786-0 xenografts treated with axitinib aloneunder low power (FIG. 6) and high power (FIG. 7), respectively.

FIG. 8 illustrates a process flow diagram for manufacturing 200 mgX4P-001 Capsules.

FIG. 9 illustrates measurements of X4P-001 200 mg capsule fills vs.theoretical capsule fill.

FIG. 10 illustrates the dissolution profile of the developed X4P-001 200mg capsules vs. the dissolution profile of the X4P-001 100 mg capsules.

FIG. 11 illustrates Western blots of 786 xenografts treated with X4P-001showed reduction in the level of HIF-2α relative to that caused byaxitinib treatment.

FIG. 12 illustrates that axitinib suppressed the micro-RNAs mir-30a andmir-30c, and the addition of X4P-001 to axitinib resulted in increasedmir-30a and mir-30c after 8 days of treatment (786-0 xenograft tumor).mir-30a and mir-30c microRNA and HIF-2α mRNA expression from tumors ofxenografts treated with axitinib+/−X4P-001. Data is presented as mir-30aor mir-30c expression relative to the mean control value (left side) andrelative HIF-2α RNA expression.

FIG. 13 illustrates that axitinib and X4P-001 together act to reduceHIF-2a expression after 8 days of treatment in 786 xenograft tumors.

FIGS. 14A-C illustrate the effect of X4P-001 treatment on 786 hypoxiccells in vitro on mir-30a and mir-30c induction and HIF-2α reduction.FIG. 14A shows a Western blot of 786 cells treated with X4P-001 for 24hours in normoxic and hypoxic (1% O₂) conditions. FIG. 14B illustratesmir-30a and mir-30c microRNA and (FIG. 14C) total HIF-2α RNA expressionfrom the same cells from FIG. 14A.

FIG. 15A illustrates Western blot results from lysates of A375 cells orA375 cells transfected with a constitutively active Stat3 construct.Cells were treated with X4P-001 for 24 h in normoxic or hypoxicconditions. FIG. 15B shows mir-30c microRNA and FIG. 15C shows total RNAexpression from the same cells from FIG. 15A. The suppression of HIF-2αand induction of mir-30a and 30c is thus dependent on Stat3 expression.Stat3 is known to be important in promoting CXCL12-mediated invasion oftumors.

FIG. 16 illustrates particle size distribution of the X4P-001 batch usedin developing the 10 mg, 25 mg, and 100 mg capsules.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

CXCR4 (C-X-C chemokine receptor type 4) is the receptor for CXCL12(C-X-C chemokine ligand type 12; also referred to as SDF-1α,stromal-derived-factor 1α). CXCL12 has potent chemotactic activity forlymphocytes and MDSCs (myeloid-derived suppressor cells), and isimportant in homing of hematopoietic stem cells to the bone marrow.CXCR4 is also expressed and active on multiple types of human cancers,including ccRCC, ovarian cancer, and melanoma, and increased expressionof CXCR4 on tumor cells has been associated with significantly decreasedoverall patient survival [3, 4, 5, 6].

Multiple observations implicate the CXCL12/CXCR4 axis in contributing tothe lack (or loss) of tumor responsiveness to angiogenesis inhibitors(also referred to as “angiogenic escape”). In animal cancer models,interference with CXCR4 function has been demonstrated to disrupt thetumor microenvironment (TME) and unmask the tumor to immune attack bymultiple mechanisms, including eliminating tumor re-vascularization[7,8], and increasing the ratio of CD8+ T cells to Treg cells [7, 9,10]. These effects result in significantly decreased tumor burden andincreased overall survival in xenograft, syngeneic, as well astransgenic, cancer models [7, 9, 8].

X4P-001 is a potent, orally bioavailable CXCR4 antagonist [11], that hasdemonstrated activity in solid and liquid tumor models [12, andunpublished data] and has previously (under the designations AMD070 andAMD11070) been in Phase 1 and 2a trials involving a total of 71 healthyvolunteers [11, 13, 14] and HIV-infected subjects [15, 16]. Thesestudies demonstrated that oral administration of up to 400 mg BID for3.5 days (healthy volunteers) and 200 mg BID for 8-10 days (healthyvolunteers and HIV patients) was well-tolerated with no pattern ofadverse events or clinically significant laboratory changes. Thesestudies also demonstrated pharmacodynamic activity, with dose- andconcentration-related changes in circulating white blood cells (WBCs);and a high volume of distribution (VL), suggesting high tissuepenetrance.

Earlier work by some of the inventors on the mechanisms of acquiredresistance to VEGF-targeted therapies, demonstrated that treatment withsunitinib treatment resulted in a marked increase in the infiltration ofrenal cell carcinoma (RCC) xenografts with CD11b+/Gr-1+ myeloid-derivedsuppressor cells (MDSC)(1). These cells have been repeatedly implicatedin the development of resistance to a diverse array of anticancertherapies, including VEGF-targeted agents (2-5). The inventors furtherobserved that the influx of MDSC, as well as the development ofsunitinib resistance, could be prevented by the concurrentadministration of the HDM2 antagonist MI-319 (Sanofi-Aventis), a drugwhose biological effects are mediated primarily through the upregulation of p53. MDSC trafficking into tumor tissue is regulated bychemokines, many of which (e.g. SDF-1 and CXCL-12) are produced inresponse to hypoxia in a HIF-dependent manner. p53 is known to directlyrepress SDF-1 transcription (6) and the inventors have shown that MI-319suppresses HIF-2 expression, suggesting that the drug may have bothdirect and indirect effects on SDF-1 expression. Based on these data,the inventors considered the possibility that MI-319 might mediate itseffects on MDSC through the suppression of chemokine (e.g. SDF-1)production. Subsequent western blot analysis of tumor lysates confirmedthis hypothesis.

These findings suggested that the ability of MI-319 to prevent sunitinibresistance might be due at least in part to the suppression of SDF-1production and MDSC recruitment. To the extent that this is the case,the inventors conceived that agents that block SDF-1/CXCR4 signalingdirectly (e.g. AMD11070) could duplicate the effects of HDM2 blockade onMDSC trafficking and prevent sunitinib resistance.

Moreover, the inventors conceived that such a result might be achievedwith comparatively little toxicity since, unlike HDM2 antagonists,CXCR4-targeted drugs would not be expected to induce cell cycle arrestin bone marrow and other normal proliferating cell populations.Accordingly, the present invention provides significant advantages intreatment outcomes utilizing the low toxicity and effects of the CXCR4inhibitor AMD11070 (X4P-001) on MDSC trafficking, differentiation, andtumor cell gene expression in RCC.

It has now been found that CXCR4 antagonism by X4P-001 providessignificant effects which may provide significant treatment benefits inpatients with advanced ccRCC and other cancers by multiple mechanisms.Administration of X4P-001 decreased recruitment of MDSC, resulting inincreased anti-tumor immune attack. Administration of X4P-001additionally sustained decreases in neoangiogenesis and tumor vascularsupply; and interferes with the autocrine effect of increased expressionby ccRCC of both CXCR4 and its only ligand, CXCL12, thereby potentiallyreducing cancer cell metastasis. Administering X4P-001, a CXCR4antagonist, sequentially (e.g. administered at the same time as separateunit dosage forms or administered as separate unit dosage forms atdifferent times separated by up to 12 h) or concurrently (e.g. takentogether) with a TKI inhibitor such as axitinib, blocks communicationbetween the tumor and the MDSC, suppresses HIF-2α expression, reducesMDSC tumor infiltration, and appreciably improves the anti-tumortreatment effect.

In the present invention, patients with advanced forms of cancer, suchas clear cell renal cell carcinoma (ccRCC) are treated with X4P-001,either as a single agent (monotherapy), or in combination with axitinib,a small molecule tyrosine kinase inhibitor (TKI) that is approved forsecond-line treatment of patients with ccRCC.

Without wishing to be bound by any particular theory, it is believedthat by combining the two medicaments, the patients' treatment outcomecan be further improved by reducing the angiogenic escape that typicallyoccurs with TKI therapy.

In some embodiments, X4P-001, or a pharmaceutically acceptable saltthereof, is administered to a patient in a fasted state.

In some embodiments, the present invention provides a method fortreating patients with cancer that presents as a solid tumor. In someembodiments, the patient has kidney cancer, renal tumor, renal carcinoma(including clear cell and papillary renal carcinoma), ovarian cancer, ormelanoma.

In some embodiments, the present invention provides a method fortreating refractory cancer in a patient in need thereof comprisingadministering X4P-001, or a pharmaceutically acceptable salt and/orcomposition thereof. In certain embodiments, the patient was previouslyadministered a protein kinase inhibitor. In some embodiments, thepatient was previously administered a VEGF-R antagonist. In someembodiments, the patient was previously administered a VEGF-R antagonistselected from axitinib (Inlyta) (Pfizer Inc., NY, USA), sorafenib(Nexavar® Bayer AG and Onyx); sunitinib (Sutent, Pfizer, New York, US);pazopanib (Votrient, GlaxoSmithKline, Research Triangle Park, US);cabozanitib (Cometriq, Exelexis, US); regorafenib (Stivarga, Bayer);lenvatinib (Lenvima, Eisai); bevacizumab (Avastin, Genentech, Inc. ofSouth San Francisco, Calif.), an anti-VEGF monoclonal antibody; andaflibercept, also known as VEGF Trap (Zaltrap; Regeneron/Sanofi). Otherkinase inhibitors/VEGF-R antagonists that are in development and may beused in the present invention include tivozanib (Aveo Pharmaecuticals,Cambridge, Mass.); vatalanib (Bayer, Novartis, Basel, Switzerland);lucitanib (Clovis Oncology); dovitinib (Novartis); CEP-11981 (Cephalon,US); linifanib (Abbott Laboratories, Abbott Park, US); PTC299 (PTCTherapeutics, South Plainfield, US); CP-547,632 (Pfizer); foretinib(Exelexis, GlaxoSmithKline); and motesanib (Amgen, Takeda).

In certain embodiments, the present invention provides a method fortreating cancer in a patient in need thereof, wherein said methodcomprises administering to said patient X4P-001 in combination with atyrosine kinase inhibitor. In certain embodiments, the X4P-001 and thetyrosine kinase inhibitor are administered simultaneously orsequentially. In certain embodiments, the tyrosine kinase inhibitor isselected from axitinib, sunitinib, sorafenib, pazopanib, cabozanitib orregorafenib. In a some embodiments of the invention, X4P-001 isadministered in combination with axitinib.

Axitinib (Inlyta® Pfizer laboratories) is a kinase inhibitor. Axitinibhas been shown to inhibit receptor tyrosine kinases including vascularendothelial growth factor receptors (VEGFR)-1, VEGFR-2, and VEGFR-3 attherapeutic plasma concentrations. These receptors are implicated inpathologic angiogenesis, tumor growth, and cancer progression.VEGF-mediated endothelial cell proliferation and survival were inhibitedby axitinib in vitro and in mouse models. Axitinib was shown to inhibittumor growth and phosphorylation of VEGFR-2 in tumor xenograft mousemodels. Axitinib has the chemical nameN-methyl-2-[3-((E)-2-pyridin-2-yl-vinyl)-1H-indazol-6-ylsulfanyl]-benzamide.The molecular formula is C₂₂H₁₈N₄OS and the molecular weight is 386.47Daltons. The chemical structure is depicted below.

Axitinib is a white to light-yellow powder with a pKa of 4.8. Thesolubility of axitinib in aqueous media over the range pH 1.1 to pH 7.8is in excess of 0.2 μg/mL. The partition coefficient (n-octanol/water)is 3.5.

Axitinib has been approved by the FDA for treatment of advanced renalcell carcinoma (RCC) after failure of one prior systemic therapy, i.e.,as second line therapy. Axitinib has been tested or mentioned as apossible treatment in other oncologic indications. Accordingly, in someembodiments of the present invention, the cancer is selected from thegroup consisting of solid tumors (including solid fibrous tumors),neoplasms (including pancreatic, kidney, colorectal, lung, breast,thyroid and stomach neoplasms), glioblastoma, hepatocellular carcinomaor liver cancer, melanoma and intraocular melanoma, prostate cancer(including castrate-resistant prostate cancer), non-small cell lungcancer, renal tumor, renal carcinoma (including clear cell and papillaryrenal carcinoma) or kidney cancer, colorectal cancer, advanced gastriccancer, malignant mesothelioma, neurofibromatosis, includingSchwannomatosis, soft tissue sarcoma, head and neck squamous cellcarcinoma, nasopharyngeal carcinoma, adenocarcinoma, neuroendocrinecarcinoma, acute myeloid leukemia, myelodysplastic syndrome,pheochromocytoma, paraganglioma, lymphoma, mantle-cell cancer,gastrointestinal-stromal tumors, or pancreatic ductal carcinoma.

In its current prescribed labeling for RCC, recommended starting oraldose of axitinib is 5 mg twice daily, approximately 12 hours apart.Depending upon individual tolerance, it is recommended that theprescribed dose of axitinib may be increased to 7 mg or 10 mg, twicedaily; or reduced to 3 mg or 2 mg twice daily.

In some embodiments, the present invention provides a method fortreating a refractory cancer in a patient in need thereof, wherein saidmethod comprises administering to said patient X4P-001 in combinationwith a tyrosine kinase inhibitor. In some embodiments, the refractorycancer is ccRCC. In some embodiments, the refractory cancer is ccRCC andthe tyrosine kinase inhibitor is axitinib.

In some embodiments, a provided method comprises administering theX4P-001, or a pharmaceutically acceptable salt thereof, is administeredto a patient in a fasted state and administering the tyrosine kinaseinhibitor to a patient in either a fasted or fed state.

In certain embodiments, the present invention provides a method fortreating cancer in a patient in need thereof, wherein said methodcomprises administering to said patient X4P-001 in combination with atyrosine kinase inhibitor, further comprising the step of obtaining abiological sample from the patient and measuring the amount of adisease-related biomarker. In some embodiments, the biological sample isa blood sample. In certain embodiments, the disease-related biomarker iscirculating CD34+ cells and/or plasma levels of soluble VEGF-R.

In certain embodiments, the present invention provides a method fortreating a refractory cancer in a patient in need thereof, wherein saidmethod comprises administering to said patient X4P-001 in combinationwith a tyrosine kinase inhibitor, further comprising the step ofobtaining a biological sample from the patient and measuring the amountof a disease-related biomarker. In some embodiments, the biologicalsample is a blood sample. In certain embodiments, the disease-relatedbiomarker is circulating CD34+ cells and/or plasma levels of solubleVEGF-R.

In certain embodiments, the present invention provides a method fortreating a refractory cancer in a patient in need thereof, wherein saidmethod comprises administering to said patient X4P-001 in combinationwith axitinib, further comprising the step of obtaining a biologicalsample from the patient and measuring the amount of a disease-relatedbiomarker. In some embodiments, the biological sample is a blood sample.In certain embodiments, the disease-related biomarker is circulatingCD34+ cells and/or plasma levels of soluble VEGF-R.

In certain embodiments, the present invention provides a method fortreating ccRCC in a patient in need thereof, wherein said methodcomprises administering to said patient X4P-001 in combination withaxitinib, further comprising the step of obtaining a biological samplefrom the patient and measuring the amount of a disease-relatedbiomarker. In some embodiments, the biological sample is a blood sample.In certain embodiments, the disease-related biomarker is circulatingCD34+ cells and/or plasma levels of soluble VEGF-R.

In other embodiments of the invention, X4P-001 is administered incombination with a VEGF antagonist. The VEGF antagonist may be anantibody to VEGF or a VEGF trap. In certain embodiments, the VEGFantagonist is selected from bevacizumab or aflibercept.

In some embodiments, the present invention provides a method of treatingcancer in a patient in need thereof, wherein said method comprisesadministering to said patient X4P-001 in combination with a tyrosinekinase inhibitor wherein the X4P-001 and the tyrosine kinase inhibitoract synergistically. One of ordinary skill in the art will appreciatethat active agents (such as X4P-001 and a tyrosine kinase inhibitor) actsynergistically when the combination of active agents results in aneffect that is greater than additive. In some embodiments, the tyrosinekinase inhibitor is axitinib.

Dosage and Formulations

X4P-001 is a CXCR4 antagonist, with molecular formula C₂₁H₂₇N₅;molecular weight 349.48 amu; appearance white to pale yellow solid;solubility: X4P-001 is freely soluble in the pH range 3.0 to 8.0 (>100mg/mL), sparingly soluble at pH 9.0 (10.7 mg/mL) and slightly soluble atpH 10.0 (2.0 mg/mL). X4P-001 is only slightly soluble in water; andmelting point of 108.9° ΔC.

The chemical structure of X4P-001 is depicted below.

In certain embodiments, the composition containing X4P-001, or apharmaceutically acceptable salt thereof, is administered orally, in anamount from about 200 mg to about 1200 mg daily. In certain embodiments,the dosage composition may be provided twice a day in divided dosage,approximately 12 hours apart. In other embodiments, the dosagecomposition may be provided once daily. The terminal half-life ofX4P-001 has been generally determined to be between about 12 to about 24hours, or approximately 14.5 hrs. Dosage for oral administration may befrom about 100 mg to about 1200 mg once or twice per day. In certainembodiments, the dosage of X4P-0001, or a pharmaceutically acceptablesalt thereof, useful in the invention is from about 200 mg to about 800mg daily. In other embodiments, the dosage of X4P-001, or apharmaceutically acceptable salt thereof, useful in the invention mayrange from about 200 mg to about 600 mg, from about 400 mg to about 800mg, from about 600 mg to about 1000 mg or from about 800 mg to about1200 mg daily.

In some embodiments, a provided method comprises administering to thepatient a pharmaceutically acceptable composition comprising X4P-001wherein the composition is formulated for oral administration. Incertain embodiments, the composition is formulated for oraladministration in the form of a tablet or a capsule. In someembodiments, the composition comprising X4P-001 is formulated for oraladministration in the form of a capsule.

In certain embodiments, a provided method comprises administering to thepatient one or more capsules comprising 10-1200 mg X4P-001 activeingredient; and one or more pharmaceutically acceptable excipients.

In certain embodiments, the present invention provides a compositioncomprising X4P-001, or a pharmaceutically acceptable salt thereof, oneor more diluents, a disintegrant, a lubricant, a flow aid, and a wettingagent. In some embodiments, the present invention provides a compositioncomprising 10-1200 mg X4P-001, or a pharmaceutically acceptable saltthereof, microcrystalline cellulose, dibasic calcium phosphatedihydrate, croscarmellose sodium, sodium stearyl fumarate, colloidalsilicon dioxide, and sodium lauryl sulfate. In some embodiments, thepresent invention provides a unit dosage form wherein said unit dosageform comprises a composition comprising 10-200 mg X4P-001, or apharmaceutically acceptable salt thereof, microcrystalline cellulose,dibasic calcium phosphate dihydrate, croscarmellose sodium, sodiumstearyl fumarate, colloidal silicon dioxide, and sodium lauryl sulfate.In certain embodiments, the present invention provides a unit dosageform comprising a composition comprising X4P-001, or a pharmaceuticallyacceptable salt thereof, present in an amount of about 10 mg, about 20mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg,about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 450 mg,about 500 mg, about 600 mg, about 700 mg, about 750 mg, about 800 mg,about 900 mg, about 1000 mg, about 1100 mg, or about 1200 mg. In someembodiments, a provided composition (or unit dosage form) isadministered to the patient once per day, twice per day, three times perday, or four times per day. In some embodiments, a provided composition(or unit dosage form) is administered to the patient once per day ortwice per day.

In some embodiments, the present invention provides a compositioncomprising:

-   -   (a) X4P-001, or a pharmaceutically acceptable salt thereof—about        30-40% by weight of the composition;    -   (b) microcrystalline cellulose—about 20-25% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate—about 30-35% by weight        of the composition;    -   (d) croscarmellose sodium—about 5-10% by weight of the        composition;    -   (e) sodium stearyl fumarate—about 0.5-2% by weight of the        composition;    -   (f) colloidal silicon dioxide—about 0.1-1.0% by weight of the        composition; and    -   (g) sodium lauryl sulfate—about 0.1-1.0% by weight of the        composition.

In some embodiments, the present invention provides a compositioncomprising:

-   -   (a) X4P-001, or a pharmaceutically acceptable salt thereof—about        37% by weight of the composition;    -   (b) microcrystalline cellulose—about 23% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate—about 32% by weight of        the composition;    -   (d) croscarmellose sodium—about 6% by weight of the composition;    -   (e) sodium stearyl fumarate—about 1% by weight of the        composition;    -   (f) colloidal silicon dioxide—about 0.3% by weight of the        composition; and    -   (g) sodium lauryl sulfate—about 0.5% by weight of the        composition.

In some embodiments, the present invention provides a compositioncomprising:

-   -   (a) X4P-001, or a pharmaceutically acceptable salt thereof—about        8-25% by weight of the composition;    -   (b) microcrystalline cellulose—about 65-85% by weight of the        composition;    -   (c) croscarmellose sodium—about 2-10% by weight of the        composition;    -   (d) sodium stearyl fumarate—about 0.1-3% by weight of the        composition; and    -   (e) colloidal silicon dioxide—about 0.05-0.7% by weight of the        composition.

In some embodiments, the present invention provides a compositioncomprising:

-   -   (a) X4P-001, or a pharmaceutically acceptable salt thereof—about        25-45% by weight of the composition;    -   (b) microcrystalline cellulose—about 10-35% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate—about 15-45% by weight        of the composition;    -   (d) croscarmellose sodium—about 2-10% by weight of the        composition;    -   (e) sodium stearyl fumarate—about 0.3-2.5% by weight of the        composition;    -   (f) colloidal silicon dioxide—about 0.05-1.2% by weight of the        composition; and    -   (g) sodium lauryl sulfate—about 0.2-1.2% by weight of the        composition.

In some embodiments, the present invention provides a compositioncomprising:

-   -   (a) X4P-001, or a pharmaceutically acceptable salt thereof—about        35-75% by weight of the composition;    -   (b) microcrystalline cellulose—about 5-28% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate—about 7-30% by weight of        the composition;    -   (d) croscarmellose sodium—about 2-10% by weight of the        composition;    -   (e) sodium stearyl fumarate—about 0.3-2.5% by weight of the        composition;    -   (f) colloidal silicon dioxide—about 0.05-1.2% by weight of the        composition; and    -   (g) sodium lauryl sulfate—about 0.2-1.2% by weight of the        composition.

In some embodiments, the present invention provides a compositionaccording to Table 1 or Table 2, below.

TABLE 1 25 mg Capsule Formulation Reference to Quantity ComponentStandard Function (mg/capsule) % w/w X4P-001 In-House Active 25.0 14.7Ingredient Microcrystalline NF Diluent 132.7 78.1 CelluloseCroscarmellose NF Disintegrant 10.2 6.0 Sodium Sodium Stearyl NFLubricant 1.7 1.0 Fumarate Colloidal USP Flow Aid 0.4 0.2 SiliconDioxide Sum Total 170.0 100.0 Hard Gelatin USP Packaging NA NA Capsules,Size 1

TABLE 2 100 mg and 200 mg Capsule Formulations 200 mg 100 mg PercentTheoretical Percent Theoretical Per Amount Per Per Amount Per CapsuleCapsule Capsule Capsule Ingredients (%) (mg) (%) (mg) X4P-001 Drug 61.5200.0 37.6 100 Substance Microcrystalline 12.9 41.93 22.9 60.9Cellulose, NF/EP (Avicel PH 101) or equivalent Dibasic Calcium 17.857.85 31.7 84.3 Phosphate Dihydrate, USP/NF Croscarmellose 6.0 19.50 6.016.0 Sodium, NF/EP (Ac-Di-Sol) Sodium Lauryl 0.5 1.625 0.5 1.3 Sulfate,NF/Ph. Eur. Colloidal Silicone 0.3 0.9750 0.3 0.8 Dioxide, NF/Ph. Eur.(Cab-O-Sil M-5P) Sodium Stearyl 1.0 3.250 1.0 2.7 Fumarate, NF (Pruv)Total Capsule Fill 100 325.0 100 266.0

In some embodiments, the present invention provides a unit dosage formcomprising a composition described above. In some embodiments, the unitdosage form is a capsule.

In as much as it may be desirable to administer a combination of activecompounds, for example, for the purpose of treating a particular diseaseor condition, it is within the scope of the present invention that twoor more pharmaceutical compositions, at least one of which contains acompound in accordance with the invention, may conveniently be combinedin the form of a kit suitable for co-administration of the compositions.Thus the kit of the invention includes two or more separatepharmaceutical compositions, at least one of which contains a compoundof the invention, and means for separately retaining said compositions,such as a container, divided bottle, or divided foil packet. An exampleof such a kit is the familiar blister pack used for the packaging oftablets, capsules and the like.

The kit of the invention is particularly suitable for administeringdifferent dosage forms, for example, oral and parenteral, foradministering the separate compositions at different dosage intervals,or for titrating the separate compositions against one another. Toassist compliance, the kit typically includes directions foradministration and may be provided with a memory aid.

The examples below explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

The contents of each document cited in the specification are hereinincorporated by reference in their entireties.

EXEMPLIFICATION Example 1: Murine Models with Human Cell Lines

The effects were examined of treatment with X4P-001 and axitinib singly,and in combination on the trafficking of MDSC and otherimmunosuppressive cell populations and on chemokine production by RCCcells.

Mice were inoculated with 786-0 and A498 RCC xenografts, the tumorspermitted to grow to ˜300 mm³, and then treatment initiated with theCXCR4 inhibitor X4P-001, axitinib, both agents in combination, or saline(control).

With each of the human cell lines, 1×107 tumor cells were implantedsubcutaneously in the flanks of 36 nude/beige mice and tumors allowed togrow to roughly 7 mm in diameter. The mice were randomly divided into 4treatment groups of 9 mice each and treated with X4P-001 (at therecommended dose), axitinib (30 mg/kg daily by gavage), both drugs, orvehicle (control). We have previously shown that MDSC tumor influx ismaximal at 7 days (not shown). Therefore, on day 7, the mice weresacrificed and the tumors were measured and immediately excised anddivided into three parts. One part of each tumor was paraffin-embeddedfor dual color immunofluorescence. Another part was mechanicallydisaggregated and treated with collagenase/DNAse to generate a singlecell suspension for flow cytometry. The third part was frozen for futurepharmacokinetic analysis. Microscope slides were made from theparaffin-embedded tumor tissue, which were stained with antibodiesagainst CD11b, Gr-1, and FAP. The number of infiltrating CD11b+/Gr-1+MDSC and FAP+ fibroblasts present in the tumor tissue was thendetermined by immunofluorescence (IF) as previously described (1).

The disaggregated tumor specimens were analyzed for CD11b+/Gr-1+ MDSCand FAP+ fibroblasts by flow cytometry. The fraction of both populationsexpressing CXCR4 were also determined. At the time the mice weresacrificed, the spleens were removed and cut in half. One part wasdisaggregated into single cell suspensions and analyzed by flowcytometry as above for MDSC. The second half was frozen for futureanalyses, such as PK analysis. Finally, a bone marrow (BM) sample wasgenerated by extruding marrow from an excised femur with a syringefilled with saline and analyzed by flow cytometry for MDSC.

Results:

Whereas either drug alone either had no (axitinib) or modest (X4P-001)effects on tumor growth, the combination of X4P-001 and axitinib hadadditive and/or synergistic antitumor effects. Specifically, combinationtreatment resulted in massive tumor cell death, with the establishedimplants actually regressing in size (See FIGS. 1A and 1B)—an effect notpreviously seen with VEGFR-targeted drugs given as single agents. IHCstaining demonstrated, as previously, that mice treated with axitinibalone had an increase in Ki-67 positive tumor cells (See FIGS. 4A and4C). This effect was not observed in mice that received both X4P-001plus axitinib (See FIGS. 4A and 4C), suggesting an anti-proliferativeeffect of the combination. Finally, the tumors from mice receivingaxitinib alone had extensive MDSC infiltration (see FIGS. 5A through 5D,whereas the tumors from mice receiving either X4P-001 alone or theaxitinib/X4P-001 combination had significantly less MDSC infiltration(see FIGS. 5A through 5D).

Suppression of miRNAs mir-30a and mir-30c and Effect on HIF-2α inXenografts:

As shown in FIG. 11, Western blots of 786 xenografts treated withX4P-001 showed reduction in the level of HIF-2α relative to that causedby axitinib treatment. Furthermore, as shown in FIGS. 12 and 13,axitinib suppressed the micro-RNAs mir-30a and mir-30c, and the additionof X4P-001 to axitinib resulted in increased mir-30a and mir-30c after 8days of treatment (786-0 xenograft tumor). mir-30a and mir-30c microRNAand HIF-2α mRNA expression from tumors of xenografts treated withaxitinib+/−X4P-001. Data is presented as mir-30a or mir-30c expressionrelative to the mean control value (left side) and relative HIF-2α RNAexpression. FIG. 13 illustrates that axitinib and X4P-001 together actto reduce HIF-2α expression after 8 days of treatment in 786 xenografttumors.

FIGS. 14A-C illustrate the effect of X4P-001 treatment on 786 hypoxiccells in vitro on mir-30a and mir-30c induction and HIF-2α reduction.FIG. 14A shows a Western blot of 786 cells treated with X4P-001 for 24hours in normoxic and hypoxic (1% O₂) conditions. FIG. 14B illustratesmir-30a and mir-30c microRNA and (FIG. 14C) total HIF-2α RNA expressionfrom the same cells from FIG. 14A.

FIG. 15A illustrates Western blot results from lysates of A375 cells orA375 cells transfected with a constitutively active Stat3 construct.Cells were treated with X4P-001 for 24 h in normoxic or hypoxicconditions. FIG. 15B shows mir-30c microRNA and FIG. 15C shows total RNAexpression from the same cells from FIG. 15A. The suppression of HIF-2αand induction of mir-30a and 30c is thus dependent on Stat3 expression.Without wishing to be bound by theory, it is believed that Stat3 isimportant in promoting CXCL-12 mediated invasion of tumors.

What these results show is that axitinib suppressed the micro-RNAsmir-30a and mir-30c, which, without wishing to be bound by theory, arebelieved to inhibit HIF-2α translation. The addition of X4P-001 toaxitinib in vivo and in hypoxic cells in vitro results in increasedmir-30a and mir-30c.

Example 2: Further Xenograft Studies

Further studies are conducted in order to determine how treatment withX4P-001 and axitinib alone or in combination affects the distribution ofMDSC and other immunosuppressive CXCR4+ cell populations (Tregs and CAF)and how CXCR4 expression by these cells affects their trafficking intumor-bearing mice. Example 1 above is repeated with additional testingof syngeneic murine RCC Renca model and 786-M1A cells, the latter ofwhich is a 786-0 variant known to express CXCR4 at extremely high levels(7). The studies with Renca cells are done as described above for thehuman cell lines except that tumors are also analyzed forCD4+/CD25bright/Foxp3+ Tregs, CD3+/CD8+ T cells in addition to MDSC andfibroblasts.

Following the procedures of Example 1, the effects of treatment withX4P-001 and axitinib on bone marrow, spleen, and tumor infiltration byCD11b+/Gr-1+ MDSC, CD4+/CD25bright/Foxp3+ Tregs, CD3+/CD8+ T cells, andFAP+ cancer-associated fibroblasts (CAF) are examined and the levels ofCXCR4 expression on these cells are determined.

Example 3: Cytokine and Chemokine Studies

The in vivo effects of treatment with X4P-001 and axitinib on chemokineproduction by RCC cells are assessed as follows:

Tumors excised from the mice undergoing treatment with X4P-001 andaxitinib in Example 1 are analyzed by RT-PCR for drug-induced changes inthe expression of M-CSF (CSF-1), CXCL1 (MGSA/gro−), CXCL2 (MIP-2/gro−),MIP-2/gro−, CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL8 (IL-8), GM-CSF, VEGF,TNF, CCL22, and CCL28. The various ELR-containing CXCL chemokines listedare known to activate CXCR2 (8), a chemokine receptor recentlyimplicated in MDSC recruitment (9). The cytokines VEGF, GM-CSF, and TNFare also thought to mediate MDSC chemotaxis into tumor tissue. CCL22 andCCL28 have been likewise implicated in the recruitment of Tregs (10,11).

Numerous chemokines and other inflammatory mediators have been shown toregulate the trafficking of MDSC into tumor tissue (9, 12, 13). Todetermine which chemokines/cytokines are responsible for the influx ofMDSC into RCC during treatment with VEGF-targeted therapies,CD11b+/Gr-1+ MDSC are isolated from the spleens of tumor-bearing miceundergoing treatment with axitinib. The MDSC are then infected with asmall pooled lentiviral shRNA library (DeCode GIPZ, Thermo Scientific)for a select group of G protein-coupled and other receptors known toregulate MDSC trafficking. The library will include shRNAs for TNFR-1and -2, IL-4R, and whole array of CXCR and CCR chemokine receptors(CXCR1-5, CCR 1-9). Several of these (e.g. CXCR-1, -2, and -4) engagechemokines known to promote MDSC recruitment (9, 12, 13).

Example 4: Pharmacokinetics Studies

In order to evaluate the pharmacokinetic properties of combined therapywith X4P-001 and axitinib, levels of X4P-001 and axitinib in blood,tumor tissue, and spleen are measured 4 hr after dosing. To measure druglevels in blood, spleen, and tumor tissue, blood is collected byventricular puncture at the time the mice are sacrificed—4 hrs after theday 7 drug dosing. The blood samples as well as the spleen and tumortissue are then subjected to PK analysis.

Example 5: Clinical Treatment Regimens

X4P-001 at a determined dose from 200 mg to 1200 mg daily isadministered orally either once daily or twice daily in divided doses.Patients are instructed about both dosing schedule and requirementsrelating to food or drink near the time of dosing.

Dosing Schedule. The first daily dose is taken in the morning and thesecond daily dose approximately 12 hours later using the followingguidelines:

-   -   Dosing should be at the same times each day±2 hr.    -   For twice daily dosing, the interval between successive doses        should not be <9 hours nor >15 hours. If the interval would        be >15 hrs, the dose should be omitted and the usual schedule        resumed at the next dose.    -   Restrictions relating to food. Absorption is impacted by food        and patients will be instructed as follows:    -   For the morning dose        -   No food or drink (except water) after midnight until the            time of dosing        -   No food or drink (except water) for 2 hours after dosing.    -   For the second daily dose, if applicable        -   No food or drink (except water) for 1 hour before dosing        -   No food or drink (except water) for 2 hours after dosing.

Axitinib is administered consistent with prescribed labelinginformation. Initial treatment with axitinib is at 5 mg orally BID inaddition to X4P-001 at the determined dose level. Administration ofaxitinib. Axitinib may be taken at the same time as axitinib.Alternatively, since axitinib has been associated with gastrointestinaladverse events and its absorption is not altered by food (see currentproduct label), patients may, with the approval of their clinician, takethe axitinib separately, following the same BID dosing scheduleguidelines noted.

Dosing of X4P-001 and/or axitinib may be adjusted by the clinician asappropriate. The dose of X4P-001 and/or axitinib may be loweredaccording to the judgment of the clinician. If a patient receivingX4P-001 in combination with axitinib experiences an adverse event atGrade >2, the dose of X4P-001 and/or axitinib may be lowered accordingto the judgment of the clinician. If a patient successfully completesthe first 4 weeks of treatment, that is, without experiencing anyadverse events greater than Grade 2, the daily dose of X4P-001 and/oraxitinib may be increased. consistent with the judgment of theclinician.

Evaluation of Response to Treatment and Disease Status

Classification of tumor response may be performed according to codifiedtumor response evaluation, according to the Response Evaluation Criteriain Solid Tumors Group (“RECIST”), as described in Therasse et al.(2000), J. National Cancer Institute, 92:205-216. Radiologic assessmentof ccRCC is accomplished by Computed Tomography (CT) with slicethickness ≤5 mm and contrast. CT is performed prior to treatment(baseline) and may be made at intervals during treatment to determinethe response.

Key Terminology

Measurable non-nodal lesions—≥10 mm in longest diameter.

Measurable nodal lesions—≥15 mm in short axis

Nonmeasurable lesions—lesions that are smaller, including those thatcannot be measured.

Measurable disease—presence of at least one measurable lesion.

Target Lesions

At baseline, four (4) measureable lesions, two (2) for each individualorgan, are identified, documented, and the appropriate diameter of eachis recorded. If measurable extra-renal lesions are present, a measurableextra-renal lesion is also identified, documented, and the appropriatediameter is recorded. Lesions are selected based on size, to berepresentative of disease, and suitable for reproducible repeatmeasurement. Target lesions may include measurable lymph nodes.

During treatment, each target lesion is assessed for Complete Response,Partial Response, Stable Disease, or Progressive Disease as follows:

Complete Response (CR)

-   -   (a) Disappearance of all non-nodal lesions, and    -   (b) Absence of pathologic lymph nodes^(a).

Partial Response (PR)

-   -   (a) ≥30% decrease from baseline in the SOD of the target lesions

Stable Disease (SD)

-   -   (a) Persisting disease that does not meet criteria for either PR        or PD Progressive Disease (PD)    -   a) ≥20% increase in the SOD of the target lesions, compared to        the smallest sum, which may be either at baseline or while on        treatment; and    -   (b) an absolute increase of ≥5 mm in the SOD.        Non-Target Lesions

All other lesions present at baseline, including pathologic nodes(defined as nodes >10 mm in short axis) should be documented(quantitative measurements are not required) so that they can beclassified on follow-up as present, absent, or unequivocal progression.

Complete Response (CR)

-   -   (a) Disappearance of all non-target lesions, and    -   (b) Absence of pathologic lymph nodes^(a).

Non-CR/Non-PD

-   -   Persistence of one or more non-target lesions

Progressive Disease (PD)

-   -   Unequivocal progression of existing non-target lesions.

[Note: ^(a)=All lymph nodes, whether or not designated target ornon-target lesions, have short axis diameter ≤10 mm.]

New Lesions

A new lesion should be unequivocal (e.g., not attributable to variationin technique); includes lesions in a location not scanned at baseline.

Pharmacokinetic Assessments

If desired, pharmacokinetic assessment of blood samples for plasmalevels of X4P-001 and axitinib may be conducted. Blood samples arecollected as scheduled. Samples are analyzed for X4P-001 concentrationusing reversed-phase high performance liquid chromatography (RP-HPLC)with MS/MS detection. The validated range of this bioanalytic method is30 to 3,000 ng/mL in plasma.

Pharmacokinetic assessment of axitinib may be accomplished usingtechniques such as described in Tortorici et al., (2011) Invest. NewDrugs 29:1370-1380, the full disclosure of which is hereby specificallyincorporated herein by reference.

Example 6: Formulation Trial Results for X4P-001

This Example summarizes pilot trial results on chosen formulation foreach of the 3 dose strengths for X4P-001. The powder blend containingAFT, fillers/diluents, a disintegrant, a glidant and a lubricant wasprepared and filled into size 1 hard gelatin capsules on an automatedcapsule filling machine. The process developed for all 3 formulationsshowed adequate flowability, acceptable weight variation and contentuniformity. All 3 formulations showed more than 90% release after 45minutes dissolution test. Amber glass bottles, each containing 30capsules, polyester coils and one desiccant pack, were individuallysealed in aluminum foil bags and placed on stability testing under 2storage conditions (2-8° C. and 25° C./60% RH).

Introduction

A total of 9 formulations (3 for each of the 3 dose strengths forX4P-001) were prepared and manually filled into size 1 hard gelatincapsules. The best capsule formulation of X4P-001 for each dose levelwas selected from three (3) formulation candidates based on 1-month R&Dstability data (Table 3). The chosen formulation for each dose level wasscaled-up for blending and capsule filling using V-blender and automatedcapsule filler, respectively.

The objectives of the pilot trial were: 1) to confirm the stability ofthe chosen formulations for X4P-001 10 mg, 25 mg and 100 mg capsulesusing a new lot of X4P-001; and 2) to collect information on scale upand the new process used for making X4P-001 capsules.

Materials and Equipment

List of Materials

X4P-001, lot #2893-A-3P

Microcrystalline Cellulose, NF, Avicel® PH-101, Lot #1155

Dibasic Calcium Phosphate Dihydrate, USP, Emcompress®, Lot #B10E

Croscarmellose Sodium, NF, Ac-Di-Sol®, Lot #T050N

Colloidal Silicon Dioxide, USP, Cab-O-Sil® M-5P, Lot #1J021

Sodium Stearyl Fumarate, NF, PRUV™, Lot #30001902

Sodium Lauryl Sulfate, NF, Lot #12810

Empty Hard Gelatin Capsules, Size 1 White Opaque, Lot #582410

60 cc Amber Glass Bottles, with a green screw-on cap

Silica Gel Desiccant Pouches, 0.5 g

Rayon Coil 12-gram/y

2×3 3-Spot Humidity Indicator Card, Lot #10018

Aluminum Foil Bags MIL-PRF-131J

List of Equipment

2-Qt. V-Blender

Bonapace In-Cap Capsule Filling Machine

Pouch Sealer

Tap Density Tester

Particle Size Analyzer (Sonic Sifter)

Experimental and Results

Selection of Formulation for the Pilot Trial

One formulation (10-E, 25-E and 100-F) was chosen for the pilot X4P-001trial at each of the 10 mg, 25 mg and 100 mg dose levels. The selectionof the formulation was mainly based on the 1-month stability profile ofthe 3 formulations for each dose under 2 storage conditions (25° C./60%RH and 2-8° C.) (Table 3). None of the formulations were stable underthe 40° C./75% RH storage condition.

Only Avicel® serves as a diluent/filler in both 10 mg and 25 mgformulations. To facilitate the capsule filling process on an automatedcapsule filler, a glidant such as colloidal silicon dioxide (Cab-O-Sil®)was explored for addition to the formulation. The trial on 2 placebobatches confirmed that the Cab-O-Sil® helps to reduce the weightvariation of capsules (Table 4). Cab-O-Sil® was also added to the 100 mgformula (100-F) that contains both Avicel® and Emcompress® to ensureadequate flow of the powder blend.

In-Process Testing

A total of 3 formulations (1 for each of the 3 dose strength forX4P-001) were prepared. The powder blend was filled into size 1 hardgelatin capsules on In-Cap Capsule Filling Machine. The weight of thefilled capsules showed about 1% in weight variability (RSD) (Table 5).

Initial Testing on Final Products

The average capsule fill weight of all batches was well within 1% of thetarget. The composite assay test results for batches #1191-10-PP,1191-25-PP and 1191-100-PP were 98.8%, 99.0% and 99.9% respectively(Table 6).

The blend uniformity of all batches was evaluated using the USP ContentUniformity test. The content uniformity of the powder blend met therequired 6% RSD (Table 6).

The dissolution test on 6 capsules from each batch was performed per USPdissolution method. All batches showed more than 99% drug release at 45minutes (Table 6).

Stability Testing

Twenty (20) amber glass bottles each containing 30 capsules, appropriateamount of polyester coils and one desiccant pack were individuallysealed in aluminum foil bags and placed on stability testing under 2storage conditions (24° C. and 25° C./60% RH) per Pilot StabilityProtocol (Table 8). One humidity indicating card was included in eachaluminum foil bag for testing the seal of each sample.

Physical Properties of X4P-001 and the Powder Blend

Particle size distribution of X4P-001 is shown in Table 9 and FIG. 16.The results of bulk density, tap density and Carr's Index are summarizedin Table 7. The physical properties of the low strength blend for the 10and 25 mg formulation were comparable to the R&D batches. However, thepowder blend of the 100 mg batch showed lower bulk and tap density dueto differences in two lots of X4P-001. The new lot is more bulky thanthe previous lot.

CONCLUSIONS

Three (3) pilot stability batches were successfully manufactured for theactive pharmaceutical ingredient (“API”), X4P-001. The current processfor all three dose levels is recommend for supporting the manufacturingof upcoming clinical batches. As used herein and in the followingTables, “API” refers to X4P-001. “API” is an abbreviation for “activepharmaceutical ingredient” that is commonly used in the art.

TABLE 3 Summary of 1-Month Stability Results on Chosen R&D Batches BatchInformation LOT NO. 1191-10-E 1191-25-E 1191-100-F Param- 25° C./ 25°C./ 25° C./ eters 2-8° C. 60% RH 2-8° C. 60% RH 2-8° C. 60% RH API 10 25100 (mg) Batch 175 175 250 Size (g) Dissolution % at 112 110 91 95 99 9645 min. Assay @ 1-month % LC 105.5 110.5 99.2 100.7 94.1 91.5 RelatedSubstances Tot. % 2.0 2.2 2.0 2.3 0.9 1.2 Area Assay @ time zero* % LC99.4 100.9 94.0 Related Substances* Tot. % 0.4 0.7 94.0 Area *The timezero data were included as reference

TABLE 4 Summary of Weight Variation Results on X4P-001 Capsules fromPilot Trials Batch Information PILOT BATCH LOT NO. Place- Place- 1191-1191- 1191- 1191- Parameters bo-1 bo-2 100-H 10-P 25-P 100-P API (mg) 00 100 10 25 100 Batch Size 200 200 200 650 650 1200 (g) Formulation API% 0.0 0.0 37.6 6.0 14.7 37.6 Avicel % 92.8 92.5 22.9 86.7 78.1 22.9Emcomp. % 0.0 0.0 31.7 0.0 0.0 31.7 Ac-Di-Sol % 6.3 6.3 6.0 6.0 6.0 6.0Cab-O-Sil % 0.00 0.25 0.23 0.25 0.24 0.30 PRUV % 1.0 1.0 1.0 1.0 1.0 1.0SLS % 0.0 0.0 0.5 0.0 0.0 0.5 TOTAL % 100.1 100.1 99.9 100.0 100.0 100.0Weight Statistics N 20 20 20 20 20 20 MIN 225.7 219.9 296.0 234.1 236.9334.1 MAX 271.3 252.8 355.9 248.5 251.0 349.6 MEAN 250.6 244.6 339.0241.9 245.0 341.0 SD 11.9 7.8 14.8 3.6 3.6 4.3 Weight Variation RSD 4.8%3.2% 4.4% 1.5% 1.5% 1.3% Wt. Var. w/o outliers N 19 19 18 N/A MEAN 251.9245.9 343.3 RSD 4.2% 2.2% 2.0%

TABLE 5 Summary of In-Process Weight Check Results on X4P-001 CapsulesLOT NO. Batch Information Parameters 1191-10-P 1191-25-P 1191-100-P API(mg) 10 25 100 Batch Size (g) 650 650 1200 Weight Statistics N 11 10 11→ MIN 239.5 239.0 336.9 MAX 245.0 249.0 344.8 MEDIAN 242.1 245.5 341.7MEAN 242.5 245.1 341.6 SD 1.7 3.0 2.3 Weight Variation RSD 0.7% 1.2%0.7% → Capsule Wt.** → 1 241.9 244.9 339.9 2 244.6 245.7 341.7 3 245.0249.0 336.9 4 241.5 245.9 344.8 5 242.1 247.3 343.3 6 242.1 239.0 342.97 240.7 242.7 341.9 8 244.6 245.3 344.3 9 242.9 242.9 341.7 10 242.1248.7 338.9 11 239.5 340.8 **Average weight of 10 capsule samples, takenevery 10 minutes during encapsulation.

TABLE 6 Summary of Time Zero Results on Pilot Stability Batches LOT NO.Batch Information Parameters 1191-10-PP 1191-25-PP 1191-100-PP API (mg)10 25 100 Batch Size 650 650 1200 (g) Target Fill 167 170 266 Wt. (mg)Wt. Var. MEAN 241.9 245.0 341.0 Capsules → RSD 1.5% 1.5% 1.3% Fill Wt.MEAN 168 171 267 Capsules → Content MEAN 96.9% 95.8% 99.9% Uniformity →RSD 2.2% 2.3% 5.2% Dissolution → % at 45 min. 99.6% 100.8% 99.7% Assay →% LC 98.8% 99.0% 99.9% Related Tot. % Area 1.4% 1.4% 1.5% Substances →

TABLE 7 Comparison of Physical Properties of Powder Blends of R&DBatches Physical Parameters Dev. Batches Pilot Batches 10 mg Batches →1191-10-E 1191-10-P Bulk Density (g/cc) 0.34 0.36 Tap Density (g/cc)0.53 0.51 Carr's Index (%) 36% 28% Mean PS (um) n/a 50    25 mg Batches→ 1191-25-E 1191-25-P Bulk Density (g/cc) 0.36 0.36 Tap Density (g/cc)0.55 0.52 Carr's Index (%) 34% 32% Mean PS (um) n/a 54    100 mg Batches→ 1191-100-F 1191-100-P Bulk Density (g/cc) 0.8  0.62 Tap Density (g/cc)1.08 0.84 Carr's Index (%) 26% 26% Mean PS (um) n/a 85   

TABLE 8 Pilot Stability Protocol PACKAGING INFORMATION: Amber 60 ccglass bottle (sealed in aluminum foil bag) Cap/Closure Type Greenplastic screw-on top Number of Bottles Packaged 15 bottles from eachbatch Number of Capsules Per Bottle 30 capsules (with polyester coilsand 1 desiccant pack) STORAGE CONDITIONS: Total Number of StorageConditions Time Points Bottles A Ambient Temperature Stability Time Zero2 + 1** B 25° C. + 2° C./60% + 1M, 3M 2 + 2** 5% RH C 2° C.-8° C. 1M, 3M2 + 2** Totals 11 TESTING TO BE COMPLETED AT EACH TIME POINT: AcceptanceNo. Test Method Performed By Criteria 1. Appearance Visual AnalyticalLab Record results 2. Content HPLC Analytical Lab USP UniformityRequirements (initial time <905> zero only) 3. Assay HPLC Analytical LabRecord results 4. Dissolution USP Analytical Lab Record resultsApparatus 2 5. Odor Olfactory Formulation Record results **Additionalback-up bottles for all conditions.

TABLE 9 Particle Size Distribution Product: X4P-001 Free Base Lot#:2893-A-3P Date: 10 Jan. 2003 Sample Wt.: 5 grams Testing Time: 5 minutes# Mesh Size Par. Size (μm) % Retained 40 425 1.3 60 250 2.5 80 180 5.8100 150 37.8 170 90 36.1 270 53 13.8 Pan <53 2.7 Sum: 100.0

Example 7: Development and Formulation of 200 me Capsule

This Example describes the development of a 200 mg strength of X4P-001Capsules and process development activities.

The formulation for X4P-001 Capsules, 100 mg was employed as a baselinefor the proposed 200 mg formulation. The goal of the formulationdevelopment activities was to obtain a higher dosage form of API with asimilar dissolution profile to the 100 mg strength and manufacture theproduct in a size 1 gelatin capsule.

A feasibility batch was manufactured using a prototype capsuleformulation (developed by Metrics) based on the excipients used in the100 mg CTM batch formulation as shown in Table 10 below. Thisfeasibility batch met all previously established drug productspecifications and displayed a drug release similar to the 100 mgstrength CTM batch (15K227). The goal of the X4P-001 Capsules, 200 mgformulation development activities was to identify an acceptable capsuleformulation to be deployed in Phase 1 clinical studies and advanced intosubsequent clinical study phases as appropriate using a scalableformulation and manufacturing process using a size 1 gelatin capsule,consistent with the current strengths (25 mg and 100 mg) of the subjectproduct line.

TABLE 10 Formulation of 200 mg and 100 mg Capsules 200 mg Strength 100mg Strength Percent Theoretical Percent Theoretical Per Amount Per PerAmount Per Capsule Capsule Capsule Capsule INGREDIENTS (%) (mg) (%) (mg)X4P-001 Drug 61.5 200.0 37.6 100.0 Substance Microcrystalline 12.9 41.9322.9 60.9 Cellulose, NF/EP (Avicel PH 101) or equivalent Dibasic Calcium17.8 57.85 31.7 84.30 Phosphate Dihydrate, USP/NF Croscarmellose 6.019.50 6.0 16.00 Sodium, NF/EP (Ac-Di-Sol) Sodium Lauryl 0.5 1.625 0.51.300 Sulfate, NF/Ph. Eur. Colloidal Silicone 0.3 0.9750 0.3 0.8000Dioxide, NF/Ph. Eur. (Cab-O-Sil M-5P) Sodium Stearyl 1.0 3.250 1.0 2.700Fumarate, NF (Pruv) Total Capsule Fill 100.0 325.0 100.0 266.0 Capsules,Empty, 1 Capsule 1 Capsule Hard Gelatin Size 1 White/White

One feasibility batch was prepared using the formulation outlined inTable 10 above. Feasibility batch manufacturing equipment included:V-shell blender (4 quart), 30 mesh hand screen, and MF-30 Manual CapsuleFiller. The manufacturing process for each batch is described below anddepicted in FIG. 8. The batch manufacture process utilized the sameprocess train as the current 100 mg strength.

1. Add the X4P-001 active ingredient to the 4 quart V-Blender.

2. Sift Avicel PH-101 and Dibasic Calcium Phosphate through a 30 meshhand screen and add to the 4 quart V-blender. Mix for 4 minutes (100rotations).

3. Sift Croscarmellose Sodium and Sodium Lauryl Sulfate through a 30mesh hand screen and add to the 4 quart V-Blender. Mix for 2 minutes (50rotations).

4. Sift Colloidal Silicon Dioxide through a 30 mesh hand screen and addto the 4 quart V-Blender. Mix for 2 minutes (50 rotations).

5. Discharge blended materials from the 4 quart V-Blender and siftthrough a 30 mesh screen. Transfer screened material back to the 4 quartV-Blender and mix for 2 minutes (50 rotations).

6. Sift Sodium Stearyl Fumarate through a 30 mesh hand screen and add tothe 4 quart V-Blender. Mix for 3 minutes (75 rotations).

7. Encapsulate the blended material using an MF-30 Manual Capsule Fillerto a target weight of 325.0 mg/capsule.

The completed final blend was encapsulated using an MF-30 Manual CapsuleFiller, filled capsule properties are presented in Table 11, below.

TABLE 11 X4P-001 Capsules, 200 mg Capsule Fill Weight Batch 15/858-034Capsule Parameter (X4P-001 Capsules, 200 mg) Tray 1 Average Weight 319.1mg Tray 2 Average Weight 320.1 mg Tray 3 Average Weight 327.6 mgIndividual Max 350.6 mg Individual Min 298.1 mg RSD (%) 3.5

Following completion of the encapsulation activities a single capsulewas filled using the MF-30 manual encapsulation to determine the maximumfill weight that could be filled into a size 1 capsule using theremaining finished blend. A fill weight of 425.0 mg was obtained duringexecution of the activity.

The conclusion of the encapsulation process development effort showedthat encapsulation is a viable operation for processing the product.

Analytical Results of X4P-001 Capsules, 200 mg Feasibility Batch.

Feasibility batch 15/858-034 was tested for Assay/Related Substances,Moisture, Dissolution, and Content Uniformity. Results of this testingare shown in FIGS. 9 and 10. The result of the assay testing was 97.4%of label claim with total impurities of 0.75% and a moisture value of3.9% w/w.

Comparison of the dissolution profile results of the 200 mg formulationcomposition compared to the 100 mg formulation CTM batch (15K227) ispresented in FIG. 10. The proposed 200 mg formulation compared favorableto the current 100 mg formulation with an f₂ similarity factor of 83.

REFERENCES

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We claim:
 1. A method for treating cancer in a patient in need thereof,wherein said method comprises administering to said patient X4P-001, ora pharmaceutically acceptable salt thereof, in combination withaxitinib, or a pharmaceutically acceptable salt thereof; wherein thecancer is refractory advanced renal cell carcinoma (RCC), the daily doseof X4P-001, or a pharmaceutically acceptable salt thereof, is from about200 mg to about 600 mg, and the daily dose of axitinib, or apharmaceutically acceptable salt thereof, is about 5 mg taken twicedaily about 12 hours apart.
 2. The method of claim 1, wherein thepatient has previously been treated with a tyrosine kinase inhibitor andwherein the patient has exhibited resistance to the tyrosine kinaseinhibitor via angiogenic escape.
 3. The method of claim 1, wherein thepatient is treated with X4P-001, or a pharmaceutically acceptable saltthereof, in an amount effective to reduce angiogenic escape, and thenthe patient receives additional treatment with axitinib, or apharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein the X4P-001, or a pharmaceutically acceptable salt thereof, andthe axitinib, or a pharmaceutically acceptable salt thereof, actsynergistically.
 5. The method of claim 1, wherein tumor cells takenfrom the patient exhibit increased expression of Ki-67 after previoustreatment with the axitinib, or a pharmaceutically acceptable saltthereof.
 6. The method of claim 1, wherein a tumor sample taken from thepatient exhibits an increase in number of myeloid-derived suppressorcells after previous treatment with the axitinib, or a pharmaceuticallyacceptable salt thereof.
 7. The method of claim 6, wherein a tumorsample taken from the patient exhibits increased infiltration area ofthe tumor by myeloid-derived suppressor cells after previous treatmentwith the axitinib, or a pharmaceutically acceptable salt thereof.
 8. Themethod of claim 1, further comprising the step of obtaining a biologicalsample from the patient and measuring the amount of a disease-relatedbiomarker.
 9. The method of claim 8, wherein the biological sample is ablood sample.
 10. The method of claim 9, wherein the disease-relatedbiomarker is circulating CD34+ cells and/or plasma levels of solubleVEGF-R.
 11. The method of claim 1, wherein the X4P-001, or apharmaceutically acceptable salt thereof, is administered orally twiceper day.
 12. The method of claim 11, wherein the daily dose of X4P-001,or a pharmaceutically acceptable salt thereof, is about 400 mg.
 13. Amethod for reducing resistance to treatment of refractory advanced renalcell carcinoma (RCC) with axitinib, or a pharmaceutically acceptablesalt thereof, at a dose of about 5 mg, taken twice daily about 12 hoursapart, in a patient receiving said treatment, said method comprisingadministering to said patient about 200 mg to about 600 mg per day ofX4P-001, or a pharmaceutically acceptable salt thereof, in an amounteffective to reduce angiogenic escape.
 14. The method of claim 13,wherein the X4P-001, or a pharmaceutically acceptable salt thereof, andthe axitinib, or a pharmaceutically acceptable salt thereof, actsynergistically.
 15. The method of claim 13, wherein the X4P-001, or apharmaceutically acceptable salt thereof, is administered orally twiceper day.
 16. The method of claim 15, wherein the daily dose of X4P-001,or a pharmaceutically acceptable salt thereof, is about 400 mg.